Atherosclerotic vascular disease is the leading cause of morbidity and mortality in the United States. Conventional treatments such as angioplasty and bypass procedures are limited in success by the development of intimal hyperplasia (IH). Over the last decade, gene therapy approaches to treating IH have been developed. We propose that one of the best candidate genes for the prevention of IH is the inducible nitric oxide synthase (iNOS) gene. iNOS produces NO which can inhibit smooth muscle cell (SMC) proliferation, promote endothelial cell (EC) regrowth, inhibit platelet and leukocyte adhesion, and promote vasodilation. These properties would be advantageous in treating IH. We used adenoviral mediated iNOS gene transfer in animal models of IH and showed dramatic reductions in IH using very low viral titers. The predominant actions of NO appear to be directed at SMC antiproliferation and EC growth. The efficacy of iNOS gene transfer has fueled an effort to develop this therapy for clinical applications. In this proposal, we will pursue three interrelated aims to further evaluate iNOS gene transfer. AIM 1: To determine the optimal conditions for therapeutic NOS gene transfer for the prevention of vascular injury induced IH. We will evaluate the impact of arginine and cofactor limitations on the efficacy of iNOS gene transfer. In addition, we will perform direct comparisons between the different NOS isoforms to see if any is NOS more efficacious. We will evaluate modified adenoviral vectors that target vascular cells for improving NOS gene delivery. AIM II: To evaluate the consequences of NOS gene therapy in a pi,, model of atherosclerosis. Human vascular disease occurs in the setting of atherosclerosis. Therefore, this AIM will focus on studying the impact of hypercholesterolemia and atherosclerosis on iNOS gene transfer efficiency and efficacy. We will also determine if a longer duration of NO production will be required in the setting of atherosclerosis to inhibit IH by a recombinant adeno-associated virus. AIM III: To further characterize the molecular mechanisms of NO- mediated vasoprotection. In this AIM, we will characterize the mechanisms by which NO inhibits SMC proliferation and promotes EC growth, focusing on the role of G-proteins, mitogen-activated protein kinase pathways, and cyclooxygenase-2. At the completion of our studies, we will have the necessary information to develop NOS gene therapy as a safe and effective treatment for vascular IH.